Amplification, detection, and automated sequencing of gibbon interleukin-2 mRNA by Thermus aquaticus DNA polymerase reverse transcription and polymerase chain reaction.
Reverse transcription-polymerase chain reaction (RT-PCR) is a gene expression assay by which messenger RNA (mRNA) production can be measured. This technique involves three steps: isolation of RNA from cells or tissues, the creation of a DNA copy of the desired message (cDNA) by viral reverse transcriptase enzymes (RT), and amplification of this DNA segment by the polymerase chain reaction (PCR) for subsequent quantitation and analysis. Here we describe a one-enzyme, one-step method combining the RT and PCR steps of conventional RT-PCR by exploiting the recently documented RT properties of Taq polymerase, the thermostable enzyme used for PCR amplification of DNA. RNA was extracted from gibbon T-cells (MLA144), reverse transcribed and amplified with oligonucleotide primers (specific for the 5' portion of a spliced interleukin-2 (IL-2) messenger RNA) by Taq polymerase. A discrete fragment of correct length for IL-2 cDNA was detected. Experiments showed that this product was RNA-dependent and specific for IL-2. This fragment was sequenced by automation employing a biotin primer-streptavidin magnetic bead protocol which confirmed its origin as processed IL-2 mRNA. The modification of the RT-PCR procedure using a thermostable enzyme speeds up reaction time and increases stringency. This method should make the diagnostic screening of cells for gene expression more efficient and practical.
Shaffer
A L
AL
Naval Medical Research Institute, Immunobiology, and Transplantation Department, Bethesda, Maryland 20814-5055.
Wojnar
W
W
Nelson
W
W
D000818:Animals; D001322:Autoanalysis; D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase; D015870:Gene Expression; D006922:Hylobates; D007376:Interleukin-2 / Q000235:genetics*; D008223:Lymphoma / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012326:RNA Splicing; D012333:RNA, Messenger / Q000096:biosynthesis*; D019914:Taq Polymerase; D013824:Thermus / Q000235:genetics*; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D007376:Interleukin-2; D012333:RNA, Messenger; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase